Nitroxides as Protectors against Oxidative Stress Summary Nitroxides are proving to have broad utility in a number of disease processes and/or conditions that represent excessive oxidative stress. The fact that nitroxides exert activity over such a range of conditions speaks to the importance of free radical reactions in tissue. Likewise, it is becoming apparent that free radicals are important in normal molecular signaling pathways and related gene expression. In collaborative studies, the effects of chronic administration of Tempol (supplemented in food) of two mouse models that exhibit neurodegeneration and/or neurological damage have been evaluated. Tempol was shown to be highly protective in an experimental autoimmune encephalomyelitis (EAE) mouse model. The EAE mouse model is an acute or chronic demyelinating autoimmune disease whose clinical manifestations of paralysis and quadriparesis that closely resemble those observed in Multiple Sclerosis (MS) patients. Pre-treatment of mice with Tempol markedly decreased paralysis. Likewise, Tempol treatment at the onset of disease decreased paralysis as well indicating that Tempol might have utility in both treating and preventing MS. We continue to search for the mechanism(s) of how long-term administration of Tempol (in the food or drinking water) results in dramatic weight reduction and a decrease in spontaneous tumor incidence in mice. Studies have been completed using an in vitro lipogenesis system (3T3L1 cells) to determine if Tempol treatment could inhibit lipogenesis. Tempol inhibited lipogenesis in this model. Further, we have found that in this system that induction of lipogenesis results in a reduction in HIF-1 alpha and an increase in prolyl hydroxylase;whereas, Tempol prevented the reduction of HIF-1 alpha and induction of prolyl hydroxylase. Studies are focused on the mechanism of this interaction, which may involve Tempol effects on the ferryl state of prolyl hydroxylase. Using this same model we have also found that Tempol prevents differiation-induced induction of PPAR-gamma and alpha along with FIAF, all of which are involved in fat storage and lipid metabolism. We are currently studying whether HIF-1 alpha expression is altered in various normal tissues in the mouse during Tempol treatment. Gene expression studies were conducted by evaluating tissue taken from age-matched control mice and mice on Tempol food supplementation for 1 month or 1 year. A number of genes were indentified in Tempol supplemented mice that were differentially up- or down-regulated in liver and brain tissue, including genes associated with glutathione metabolism (up-regulated). Genes associated with fat synthesis and storage were found to be modulated by Tempol. The gene array study suggested that hypoxic related genes were also modulated by Tempol. Finally, another approach toward elucidating a mechanism for Tempol effects on animal weight is by evaluating metabolic products in the urine. The hypothesis to be tested is that mice consuming Tempol in their food will have a different metabolic profile than control mice. We have collected urine samples from control and Tempol supplemented mice for metabolomics evaluation. Preliminary data show that there are numerous break-down metabolic products of Tempol. Data evaluation is currently ongoing.